HDAC inhibition decreases p53 levels similarly to TRRAP silencing. (A) Impact of different small-molecule inhibitors on p53 levels. Lymphoma cell lines were treated with 0.1% DMSO or the indicated inhibitors. 24 hours after treatment, cells were subjected to p53 flow cytometry. wtp53 cell lines were additionally treated with 25 µM etoposide for 5 hours before harvest to stabilize p53. The p53 mutation status is specified for each cell line (green, wtp53; red, mutp53). Values in heatmap denote log2 fold changes of p53 MFI normalized to DMSO-treated or etoposide-treated cells, respectively. Values in fluorescence-activated cell sorter (FACS) plots denote the ratio of the p53 MFI between vorinostat-treated (red) and DMSO-treated cells (gray). For cell lines with a bimodal p53 level, gates and the corresponding percentages for the p53-low and p53-high population are indicated (10 µM Nutlin-3a, MDM2 inhibitor; 5 µM 17-AAG, Hsp90 inhibitor; 5 µM MG-132, proteasome inhibitor; 10 µM chloroquine, autophagy inhibitor; 5 µM vorinostat, HDAC inhibitor). (B) Effect of vorinostat on mutp53 expression and evaluation of MDM2-dependency. Namalwa cells were treated with DMSO (0.1%, gray), vorinostat (5 µM, red), or with a combination of vorinostat and Nutlin-3a (10 µM, blue) for 24 hours before they were harvested. mRNA expression values (mean + SD, n = 2) determined by qRT-PCR and normalized to GAPDH and to DMSO-treated cells (left). p53 flow cytometry (right). Values denote p53 MFI normalized to DMSO-treated cells. (C) p53 flow cytometry of Namalwa cells after treatment with 0.1% DMSO (gray) or different HDAC inhibitors (red). The target proteins of the different inhibitors are indicated. Values denote p53 MFI normalized to DMSO-treated cells (7.5 µM tubacin; 7.5 µM tubastatin A; 10 µM PCI-34051; 500 nM entinostat; 500 nM mocetinostat). (D) Correlation of the impact of TRRAP silencing (8 days after transduction) and Vorinostat treatment (5 µM, 24 hours) on mutp53 levels determined by flow cytometry. Values denote p53 MFIs normalized to either untransduced or DMSO-treated cells, respectively. FACS plots in parts reproduced from panel A. The Pearson correlation coefficient (r) and the corresponding P value are indicated. (E) Gene set enrichment analysis of differentially expressed genes after TRRAP knockdown (shRNA #233) in mutp53 Namalwa, wtp53 Seraphina, and p53^KO Seraphina cells (compared with nontargeting control). Cells were selected with puromycin for 48 hours and harvested 6 to 8 days after transduction. Analysis was performed using PAGE with the MSigDB gene set collection “c2” filtered for the term “HDAC.” Only gene sets that were significantly altered in at least 1 cell line are shown (adjusted P < .05), and nonsignificant enrichments are shown in gray. HDAC1/2/3-related pathways are highlighted.⁵⁰  (F) Impact of HDAC inhibition and TRRAP silencing on mutp53 acetylation. Namalwa cells were treated with DMSO (0.05%) or different HDAC inhibitors before they were subjected to p53 or control (IgG) immunoprecipitation (IP). Silencing experiments were performed using Namalwa cells with IPTG-inducible shRNAs against TRRAP (shRNA #233) or a nontargeting control (NT). Cells were harvested 18 hours after drug exposure or 4 days after shRNA induction and were treated with the deacetylase inhibitors trichostatin A (1 µM) and nicotinamide (5 mM) for the last 4 hours of culture to enrich for acetylated proteins. Protein expression was determined by western blot and normalized to GAPDH (input samples). Acetylation (Ac) of p53 was detected using an antiacetylated lysine antibody and normalized to total p53 (IP samples) and to cells transduced with the NT or to DMSO-treated cells, respectively. Asterisk indicates an unspecific band. Ac-BSA, acetylated bovine serum albumin, positive control; vorinostat, 1 μM; entinostat, 500 nM; mocetinostat, 500 nM.